A method for separating according to mass a mixture of macromolecules or small particles suspended in a fluid, 3. Experiments in a centrifugal fluid.

Our first paper' presented the theory of a method for separating a mixture of particles according to mass. The second2 reported the results obtained with a gravitational device in the effective mass range 10-14 to 10-'3 gm. Here we describe experiments designed to test the feasibility of separating particles with effective masses as small as 10-18 gm. In the gravity experiments particles are added at one end of a thin layer of fluid contained in a long rectangular trough. They settle through the liquid and form Boltzmann distributions near the bottom. The scale height of such a distribution depends on what we have called the effective mass of the particle, the mass of the particle less the mass of the fluid it displaces. When the fluid is made to flow along the trough, light particles travel with a higher average velocity than heavy particles because their distributions extend into the more rapidly moving parts of the fluid. The speed at which an individual particle is transported varies as the particle diffuses up and down in the liquid. If these excursions occur frequently enough, the average velocity of each particle of a given kind is nearly the same, and particles of a given kind travel as a group. This averaging is effective only if the time for diffusion through one scale height is short compared to the duration of the experiment. The diffusion time increases rapidly with decreasing effective mass.3 Therefore, light particles cannot be separated in a reasonable length of time unless the scale height is reduced. This can be done by using a centrifugal field. For this purpose we constructed a special centrifuge with a long, hollow cylindrical rotor turning about a vertical axis (Fig. 1). Liquid added at the top of the spinning rotor collects as a layer on the inside wall until its depth becomes equal to the height of a shallow barrier at the bottom. Here "depth" and "height" refer to distances measured radially, i.e., horizontally. If more liquid is added, an equal volume spills out over the barrier. The fluid layer, held against the inner wall of the rotor by the centrifugal field, thus has a depth fixed by the barrier height. The particles to be separated are added to this layer at the top of the rotor. They sediment radially, forming Boltzmann distributions in the centrifugal field. Liquid is then injected continuously so that the fluid moves slowly down the rotor